Abstract
Not all cells behave uniformly after treatment in tissue engineering studies. In fact, some treated cells display no signs of treatment or show unique characteristics not consistent with other treated cells. What if the “unique” cells could be isolated from a treated population, and further studied? Photo-convertible reporter proteins, such as Dendra2, allow for the ability to selectively identify unique cells with a secondary label within a primary labeled treated population. In the current study, select cells were identified and labeled through photo-conversion of Dendra2-transfected human Wharton’s Jelly cells (hWJCs) for the first time. Robust photo-conversion of green-to-red fluorescence was achieved consistently in arbitrarily selected cells, allowing for precise cell identification of select hWJCs. The current study demonstrates a method that offers investigators the opportunity to selectively label and identify unique cells within a treated population for further study or isolation from the treatment population. Photo-convertible reporter proteins, such as Dendra2, offer the ability over non-photo-convertible reporter proteins, such as green fluorescent protein, to analyze unique individual cells within a treated population, which allows investigators to gain more meaningful information on how a treatment affects all cells within a target population.
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Acknowledgments
This project was funded by the NIH (R01 AR056347) and the State of Kansas. We would like to acknowledge the efforts of the nursing staff at Lawrence Memorial Hospital (Lawrence, KS) for assisting us in obtaining human umbilical cords with the informed consent of patients. Furthermore, we would like to acknowledge the efforts of Peggy Keefe, Austin Smith, and Mackenzie Bloom for their assistance on this project.
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Adam J. Mellott, Heather E. Shinogle, David S. Moore, and Michael S. Detamore declare no conflicts of interest.
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The authors have no financial or intellectual property conflicts to disclose.
Ethical Standards
All human subjects research were carried out in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study. No animal studies were carried out by the authors for this article.
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Associate Editor Alyssa Panitch oversaw the review of this article.
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12195_2014_365_MOESM1_ESM.mov
Supplementary Fig. 1. Time-lapse video of full photo-conversion of hWJCs. All hWJCs converted from green to red fluorescence with gentle exposure to UV light. Duration = 00:58. Scale Bar = 100 μm. (MOV 1468 kb)
12195_2014_365_MOESM2_ESM.mov
Supplementary Fig. 2. Time-lapse video of isolated photo-conversion of hWJC. A single hWJC was photo-converted from green to red fluorescence with targeted UV light exposure, while surrounding cells did not photo-convert. Duration = 01:41. Scale Bar = 100 μm. (MOV 12189 kb)
12195_2014_365_MOESM3_ESM.avi
Supplementary Fig. 3. 48 h time-lapse video of the movement of photo-converted hWJCs. Select hWJCs were photo-converted, and were tracked over the course of 48 h. No cells that came into contact with the photo-converted cells displayed any signs of photo-conversion. Duration = 00:30. Scale Bar = 100 μm. (AVI 4655 kb)
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Mellott, A.J., Shinogle, H.E., Moore, D.S. et al. Fluorescent Photo-conversion: A Second Chance to Label Unique Cells. Cel. Mol. Bioeng. 8, 187–196 (2015). https://doi.org/10.1007/s12195-014-0365-4
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DOI: https://doi.org/10.1007/s12195-014-0365-4